基于肺与大肠相表里，研究大承气汤调控NETosis保护重症急性胰腺炎胰-肠-肺轴炎性损伤的机制

NETosis is a process of neutrophil-specific cell death characterized by the release of web-like structure referred to as neutrophil extracellular traps. It induces pancreas-intestine-lung inflammatory injury of severe acute pancreatitis (SAP) , which is the main cause of early death of SAP. Da-Cheng-Qi Decoction (DCQD) can improve gastrointestinal motility and relieve the lung injury, but the targets and mechanisms of DCQD inhibiting inflammatory reaction by regulating NETosis to protect pancreas-intestine-lung injury are still unclear. Our preliminary trials showed that DCQD improved gastrointestinal motility and relieved the lung injury; cell-free DNA was increased in SAP patients compared to mild acute pancreatitis patients. Based on our preliminary results, the potential role of NETosis in pancreas-intestine-lung inflammatory injury, and guided by “lung and large intestine are internal externally related” theory, we hypothesized that DCQT regulates NETosis to inhibit inflammatory reaction and then protect pancreas-intestine-lung inflammatory in SAP. Our project is ① To explore the mechanisms of NETosis in experimental SAP pancreas-intestine-lung inflammatory injury; ② To verify the protective effects and mechanisms of DCQT regulating NETosis to pancreas-intestine-lung from inflammatory injury in SAP rats induced by sodium taurocholate/arginine; ③ To verify the protective effects and mechanisms of DCQD in the injury of pancreatic acinar cells, lung epithelium cells and intestinal epithelium cells caused by neutrophil NETosis. The aim of this project is to illuminate the core target and the mechanisms of DCQD in inhibiting SAP inflammatory and preventing pancreas-intestine-lung injury inflammatory injury. The results will further prove the “lung and large intestine are internal externally related” theory and provide the experimental evidence to clinical practice.

NETosis是中性粒细胞释放胞外诱捕网而死亡的过程，其介导的胰肠肺炎性损伤是重症急性胰腺炎（SAP）早期主要死因。大承气汤能改善胃肠动力以减轻肺损伤，但其调控NETosis-抑制炎症以保护胰肠肺损伤的靶点和机理不清。基于预实验结果①大承气汤改善SAP胃肠动力-减轻肺损伤②SAP患者血游离DNA升高，结合NETosis在胰肠肺轴炎性损伤的潜在作用和“肺与大肠相表里”，假设：大承气汤调控NETosis—抑制炎症以防治SAP胰-肠-肺轴炎性损伤。拟探索①NETosis介导SAP胰肠肺轴炎性损伤的机理②大承气汤调控NETosis以保护牛黄胆酸/精氨酸诱导SAP胰肠肺轴炎性损伤的效应及机制③离体研究验证大承气汤调控NETosis以保护胰腺腺泡、肺及肠上皮细胞损伤机制。最终阐明大承气汤调控NETosis-抑制SAP炎症以防治胰肠肺炎性损伤的核心靶点和机理，反证肺与大肠相表里，为临床应用提供实验依据。

目的：从体内和体外实验探究大承气汤调控胰-肠-肺NETosis轴保护重症急性胰腺炎大鼠急性肺损伤的机理。前期发现大承气汤的有效成分大黄素在SAP大鼠肺组织中浓度最高，进一步探究大黄素能否代替母方发挥同样的抑制胰-肠-肺NETosis轴保护重症急性胰腺炎大鼠急性肺损伤的作用及机制。.方法：分别以精氨酸腹腔注射和牛黄胆酸钠逆行胰胆管注射法建立两种SAP模型，并随机对照组、模型组（精氨酸/牛磺胆酸钠）、DNase I组、大承气汤组，电镜和免疫荧光法检测各组织NETosis结构，HE观察各组织病理损伤，ELISA检测各组织炎症因子和ROS的产生；提取大鼠中性粒细胞，随机分为PMA、LPS、TNF-a/IL-6混合物和大黄素组，电镜和免疫荧光法检测各组细胞的NETosis产生情况，WB检测各组细胞PMN的信号通路蛋白；将PMA诱导形成的NETosis随机与胰腺腺泡细胞、I型肺泡上皮细胞、结肠上皮细胞共孵育，并设置大黄素治疗对照组，收集细胞培养上清液和细胞，运用ELISA进行相关蛋白酶指标检测，和Western-blot检测上皮细胞屏障蛋白和肺组织炎症指标。另外，在既往肺泡上皮细胞分离纯化的方案中，加用谷氨酸将培养基PH值调节到6.0-6.5左右进行条件性培养。.结果：大承气汤可以有效阻断牛磺胆酸钠SAP大鼠胰-肠-肺轴中性粒细胞NETosis数量、抑制胰酶分泌、保护肠粘膜屏障，最终改善SAP相关肺损伤；炎性介质LPS、TNF-α和IL-6的复合物能诱导中性粒细胞产生NETosis，而大黄素能抑制PMA和炎性相关产物诱导的NETosis形成、PMN的信号通路蛋白的表达和ROS的释放；相比PMN，NETosis能引起胰腺腺泡细胞、肺泡上皮细胞及结肠上皮细胞更严重的损伤，相比之下，大黄素能够通过抑制MPO的表达从而减轻NETosis导致的细胞炎症损伤。加入谷氨酸调整后得到PH为6.5左右的培养基进行培养，可抑制成纤维细胞的生长，使AEC的纯度达到99%。.结论：大承气汤可以通过阻断胰-肠-肺中性粒细胞NETosis改善SAP相关性ALI；其有效成分大黄素能抑制胰腺腺泡细胞、肺泡上皮细胞及结肠上皮细胞的NETosis的形成、改善胰-肠-肺炎症环境。另外，发明了一种肺泡I型上皮细胞的选择性培养及其分离纯化方法。